Nanopipette apparatus for manipulating cells

ABSTRACT

Disclosed herein are methods and systems for controlled ejection of desired material onto surfaces including in single cells using nanopipettes, as well as ejection onto and into cells. Some embodiments are directed to a method and system comprising nanopipettes combined with an xyz controller for depositing a user defined pattern on an arbitrary substrate for the purpose of controlled cell adhesion and growth. Alternate embodiments are directed to a method and system comprising nanopipettes combined with an xyz controller and electronic control of a voltage differential in a bore of the nanopipette electroosmotically injecting material into a cell in a high-throughput manner and with minimal damage to the cell. Yet other embodiments are directed to method and system comprising functionalized nanopipettes combined with scanning ion conductance microscopy for studying molecular interactions and detection of biomolecules inside a single living cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 61/448,998 filed on Mar. 3, 2011, which is herebyincorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT DISK

None.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of nanodevices andnanosensors for single cell injection, patterning and detection ofbiomolecules.

2. Related Art

Presented below is background information on certain aspects of thepresent invention as they may relate to technical features referred toin the detailed description, but not necessarily described in detail.That is, individual parts or methods used in the present invention maybe described in greater detail in the materials discussed below, whichmaterials may provide further guidance to those skilled in the art formaking or using certain aspects of the present invention as claimed. Thediscussion below should not be construed as an admission as to therelevance of the information to any claims herein or the prior arteffect of the material described.

Nanopipette technology has been shown to be a capable platform for manyapplications. Actis et al., (including two of the present inventors)developed a sensing platform, named STING (“Signal Transduction by IonNano Gating”), where the very tip of a quartz nanopipette isfunctionalized with chemical or biological receptors (Actis, P., O.Jejelowo, and N. Pourmand, Ultrasensitive mycotoxin detection by STINGsensors. Biosensors & Bioelectronics, 2010. 26(2): p. 333-337). Thenanometer scale opening at the tip creates a region that is sensitive toanalyte binding with the attached receptors. Additionally, Rodolfa, etal., have shown that nanopipettes can be used for controlled depositiononto functionalized surfaces (Rodolfa, K. T., et al., Two-componentgraded deposition of biomolecules with a double-barreled nanopipette.Angewandte Chemie-International Edition, 2005. 44(42): p. 6854-6859).Further work has demonstrated deposition of material onto a surface inan inorganic solvent (Rodolfa, K. T., et al., Nanoscale pipetting forcontrolled chemistry in small arrayed water droplets using adouble-barrel pipet. Nano Letters, 2006. 6(2): p. 6). Individualmolecules were delivered to cells' plasma membranes using nanopipettes(Bruckbauer, A., et al., Nanopipette delivery of individual molecules tocellular compartments for single-molecule fluorescence tracking.Biophysical Journal, 2007. 93: p. 3120-3131).

Laforge et al. developed an electrochemical attosyringe, based on ananopipette that delivers liquid by applying a voltage across theliquid/liquid interface formed at the nanopipette opening (Laforge, F.O., et al., Electrochemical attosyringe. Proceedings of the NationalAcademy of Sciences of the United States of America, 2007. 104(29): p.11895-11900). The resulting force is sufficiently strong to induce theflow of liquid into/out of the pipette. They have successfully used thiseffect to deliver femtoliters of aqueous solution into mammalian cellsin culture. Carbon nanopipettes have shown efficacy in cell injection aswell. Pressure driven injection of fluorescent dyes into oral squamouscarcinoma cells was demonstrated by Schrlau, et. al. (Carbonnanopipettes for cell probes and intracellular injection.Nanotechnology, 2008: p. 015101-1-4).

Cell Patterning

Controlling cell attachment and growth has emerged as an important topicin biological disciplines from neuroscience to stem cell research(James, C. D., et al., Aligned microcontact printing of micrometer-scalepoly-L-lysine structures for controlled growth of cultured neurons onplanar microelectrode arrays. Ieee Transactions on BiomedicalEngineering, 2000. 47(1): p. 17-21; Welle, A., et al., Photo-chemicallypatterned polymer surfaces for controlled PC-12 adhesion and neuriteguidance. Journal of Neuroscience Methods, 2005. 142(2): p. 243-250). Bycontrolling where and how cells grow and mature, specificcharacteristics can be induced during cell growth. For instance, it hasbeen shown by Oliva, et. al. (Patterning axonal guidance molecules usinga novel strategy for microcontact printing. Neurochemical Research,2003. 28(11): p. 1639-1648), that neuron differentiation can becontrolled by chemical cues, allowing the direction of axon growth to bepredetermined and customized for specific experiments. Similarly, inkjetprinting has been shown to be capable of controlling neural stem celldifferentiation (Ilkhanizadeh, S., et al., Inkjet printing ofmacromolecules on hydrogels to steer neural stem cell differentiation.Biomaterials, 2007. 28(27): p. 3936-3943).

Cell patterning has been investigated using a multitude of methods. Byleveraging the advancements made in semiconductor fabrication technologypatterns with feature sizes small enough to guide single cells has beenmade possible. The most widely used cell patterning technique ismicrocontact printing (μCP), whereby a master mold is fabricated usingtraditional photolithography, and an elastomeric stamp is created fromthe master. The stamp can then be inked with biomolecules and thepattern can be applied to an arbitrary substrate (Wilbur, J. L., et al.,Microfabrication by microcontact printing of self-assembled monolayers.Advanced Materials, 1994. 6(7-8): p. 600-604). This method has seen widesuccess in controlling cell growth, particularly in the arena ofcontrolled neural growth (Park, T. H. and M. L. Shuler, Integration ofcell culture and microfabrication technology. Biotechnology Progress,2003. 19(2): p. 243-253). Photolithography itself can be used forpatterning as well. By exposing a substrate to radiation through a mask,surface chemistry can be modified, thus allowing specific attachmentmolecules to be placed in the appropriate regions (Welle, A., et al.,Photo-chemically patterned polymer surfaces for controlled PC-12adhesion and neurite guidance. Journal of Neuroscience Methods, 2005.142(2): p. 243-250). Topographic cues have been shown to control cellgrowth as well. By changing the surface roughness that cells interfacewith cells adhesion can be controlled. The combination of chemical andtopographical attachment cues has even been demonstrated as leading toimproved cell differentiation (for example, Stenger, D. A., et al.,Microlithographic determination of axonal/dendritic polarity in culturedhippocampal neurons. Journal of Neuroscience Methods, 1998. 82(2): p.167-173). The drawback to these methods is that once the pattern hasbeen designed and fabricated it cannot be changed without designing anew mask and restarting the entire process from scratch. Some methodshave been developed for patterning that do not rely on standardsemiconductor fabrication technology, and therefore are not limited bythe fabrication process. Gustaysson, et al., (Neurite guidance onprotein micropatterns generated by a piezoelectric microdispenser.Biomaterials, 2007. 28(6): p. 1141-1151) demonstrated a piezo actuatedmicrodispenser capable of depositing 100 μL droplets with a precision of6-8 μm. Dip pen and fountain pen lithography allows spot sizes as smallas 40 nm to be deposited in an arbitrary user defined pattern as shownby Schmidt, R. C. and K. E. Healy (Controlling biological interfaces onthe nanometer length scale) in Journal of Biomedical Materials ResearchPart A, 2009. 90A(4): p. 1252-1261. However this technique is limited todeposition of only a single pattern at a given time.

By using quartz nanopipette technology described below, the patterndeposited on a substrate is computer controlled and can thus be modifiedat any time by the user, and is capable of easily depositing multiplepatterns that are registered relative to each other. The pattern can beused to accomplish cell attachment, as shown in FIG. 2, to a substrate216, where a cell 212 is attached to a point on the substrate where anadhesion material has been deposited.

Cell Injection

Methods for cell injection have historically used a pulled glassmicropipette. Traditional micropipettes suffer from several drawbacksincluding large size relative to typical cells, low cell viability, lackof feedback and the requirement of a skilled operator (Pillarisetti, A.,et al., Evaluating the effect of force feedback in cell injection. IeeeTransactions on Automation Science and Engineering, 2007. 4(3): p.322-331; Stephens, D. J. and R. Pepperkok, The many ways to cross theplasma membrane. Proceedings of the National Academy of Sciences of theUnited States of America, 2001. 98(8): p. 4295-4298).

A multitude of other methods for cell injection have been developed toalleviate these drawbacks. Methods such as electroporation, and the useof the pore forming toxin streptolysin-O (SL-O) have been developed forpassive transfer of material into the cell (Wang, M. Y., et al.,Single-cell electroporation. Analytical and Bioanalytical Chemistry,2010. 397(8): p. 3235-3248; Knight, D. E. and M. C. Scrutton, Gainingaccess to the cytosol—the technique and some applications ofelectropermeabilization. Biochemical Journal, 1986. 234(3): p. 497-506;Giles, R. V., et al., Selecting optimal oligonucleotide composition formaximal antisense effect following streptolysin O-mediated delivery intohuman leukaemia cells. Nucleic Acids Research, 1998. 26(7): p.1567-1575). Electroporation has been demonstrated to induce transientpermeability in the cell membrane by application of high voltage, afterwhich material can diffuse in. Cells have shown the ability to heal SL-Olesions under certain circumstances. In both cases, stress is introducedto the cell, and in the case SL-O lesions uptake is limited to ˜100 kDa.

Direct methods of cell injection have been demonstrated using otherunrelated nanofabricated structures. A nanoneedle, fabricated on AFMtips and coated with DNA was inserted into a single cell. Injection wasaccomplished by diffusion of DNA from the nanoneedle (Sung-Woong, H., etal., High-efficiency DNA injection into a single human mesenchymal stemcell using a nanoneedle and atomic force microscopy. Nanomedicine:Nanotechnology, Biology and Medicine, 2008: p. 215-25). Similarly,quantum dots were delivered by cleavage of disulfide bonds linking thedots to a nanoneedle within a cell (Chen, X., et al., A cellnanoinjector based on carbon nanotubes. Proceedings of the NationalAcademy of Sciences of the United States of America, 2007. 104(20): p.8218-8222).

Single Cell In Vivo Detection of Intracellular Molecular Species

Several methods allowing intracellular recording have been developed.Kopelman and coworkers (Tan, W., et al., Submicrometer intracellularchemical optical fiber sensors. Science 258, 778-781,doi:10.1126/science.1439785, 1992; Barker, S. L. R., et al., CellularApplications of a Sensitive and Selective Fiber-Optic Nitric OxideBiosensor Based on a Dye-Labeled Heme Domain of Soluble GuanylateCyclase. Analytical Chemistry 71, 2071-2075, doi:10.1021/ac9901081,1999) pioneered the application of chemical modified tapered opticalfiber for the extracellular monitoring of pH and nitric oxide. Vo-Dihnand coworkers reported the analytical application of antibody modifiedoptical fiber for measurement of a fluorescent analyte in a single cell(Vo-Dinh, T., et al., Antibody-based nanoprobe for measurement of afluorescent analyte in a single cell. Nat Biotech 18: 764-767, 2000).One of the advantages of employing tapered optical fiber relies on highspatial resolution achievable using near field scanning opticalmicroscopy. Sensors must be carefully manipulated under a microscope toavoid damage to cells. In addition to these physical constraints, theselective detection of biomolecules by affinity methods is itselfchallenging due to the many interfering species inside the cytoplasm.More recently an optical fiber nanobiosensor was constructed to detect acancer biomarker into a single cell through an enzymatic sandwichimmunoassay (Zheng, X. T. & Li, C. M. Single living cell detection oftelomerase over-expression for cancer detection by an optical fibernanobiosensor. Biosensors and Bioelectronics 25: 1548-1552, 2010).Electrical detection methods are known to be more suitable than othermethods due to improved durability, sensitivity, rapid response, andintegration with other device components. Even so, electrical-basedsensors for intracellular measurements face many challenges.Microelectrodes, are usually large enough to damage typical mammaliancells (5 to 10 μm), and procedures are often limited to measurements inoocytes and embryos, which are at least ten times larger. Recently,microelectrodes protruding inside cells were employed to measuresub-threshold synaptic potentials by Hai, A., et al. (In-cell recordingsby extracellular microelectrodes.) in Nature Methods (2010) 7, 200-202.Despite successful intracellular cation and pH sensors usingmicroelectrodes coated with ion-selective membranes by Bakker, E. &Pretsch, E. (Nanoscale potentiometry.) in TrAC Trends in AnalyticalChemistry 27, 612-618, the intracellular electrical detection ofbiomolecules remains elusive. Lieber et collaborators developed a novelapproach where a nanoscale field effect transistor (nanoFET) modifiedwith phospholipid bilayers was able penetrate a single cell and recordintracellular potentials (Tian, B. et al. Three-Dimensional, FlexibleNanoscale Field-Effect Transistors as Localized Bioprobes. Science 329,830-834, doi:10.1126/science.1192033, 2010). However no electricalsensors were employed to detect biomolecular interaction inside a singlecell.

STING (“Signal transduction by ion nano-gating”) technology has alsobeen shown capable of detecting DNA, proteins and mycotoxins in a sample(Fu, Y., et al., Nanopore DNA sensors based on dendrimer-modifiednanopipettes. Chem Commun (Camb), (2009), 4877-4879,doi:10.1039/b910511e; Umehara, S., et al., Label free biosensing withfunctionalized nanopipette probes. Proceedings of the National Academyof Sciences (2009) 106, 4611-4616, doi:10.1073/pnas.0900306106; Actis,P., et al., Ultrasensitive mycotoxin detection by STING sensors.Biosensors and Bioelectronics (2010)26, 333-337). Based on afunctionalized quartz nanopipette, STING technology does not require anynanofabrication facility; each probe can be easily and inexpensivelytailored at the bench. Receptor molecules can be incorporated using wellestablished surface chemistries. Besides biosensing, nanopipetteplatform were used to study single molecule biophysics, controlleddelivery inside individual cells, and to image cells at the nanoscale(Clarke, R. W., et al., Trapping of proteins under physiologicalconditions in a nanopipette. Angew Chem Int Ed Engl (2005), 44,3747-3750, doi:10.1002/anie.200500196; Laforge, F. O., et al.,Electrochemical attosyringe. Proceedings of the National Academy ofSciences (2007), 104, 11895-11900, doi:10.1073/pnas.0705102104;Klenerman, D. & Korchev, Y. Potential biomedical applications of thescanned nanopipette. Nanomedicine (Lond) (2006), 1, 107-114,doi:10.2217/17435889.1.1.107). Vitol and coworkers developed a SERSactive carbon nanopipette for intracellular analysis (Singhal, R. et al.Small diameter carbon nanopipettes. Nanotechnology, (2010), 015304).SERS functionality was added by incorporating gold nanoparticles on theouter surface pipette tip. SERS spectra obtained with the nanopipetteinserted within the nucleus show typical features associated with DNA.

However, there remains a need in the art for a nanopipette biosensorthat can operate within a living cell.

SPECIFIC PATENTS AND PUBLICATIONS

Karhanek et al. in US Patent Application Publication 2010/0072080,published on Mar. 25, 2010, disclose methods and devices comprising afunctionalized nanopipette for biomolecular detection, including ofpeptides and proteins.

Umehara et al. in Proceedings of the National Academy of Sciences, vol106, pages 4611-4616, Mar. 24, 2009, disclose a label-free, real-timeprotein assay using functionalized nanopipette electrodes.

Ying, Liming in Biochemical Society Transactions, vol 37, pages 702-706,2009, reviews nanopipettes and their use in nanosensing andnanomanipulation of ions, molecules (including biomolecules) and cells.

Actis, P., et al. in Bioanalytical Reviews 1, 177-185,doi:10.1007/s12566-010-0013-y (2010) review applications of nanopipettetechnology as electrochemical biosensors for nucleic acids and smallproteins.

Hansma et al. U.S. Pat. No. 4,924,091 issued May 8, 1990, entitled“Scanning Ion Conductance Microscope,” describes a scanning ionconductance microscope, SICM, which can image the topography of softnon-conducting surfaces covered with electrolytes by maintaining amicropipette probe at a constant conductance distance from the surface.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under Contract NumberNCC9-165 and NNX08BA47A awarded by the National Aeronautics and SpaceAdministration (NASA), Contract Number P01-HG000205 awarded by theNational Institutes of Health, Contract Number NNX09AQ44A awarded byNASA and under Contract Number U54CA143803 awarded by the NationalCancer Institute. The Government has certain rights in this invention.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

In certain aspects, the present invention pertains to an apparatus fordepositing onto a substrate a liquid in a predefined location havingsubmicron features, comprising: (a) a multi-barreled nanopipette,wherein two barrels have inside them a first and second electrode, saidfirst electrode in a barrel adapted to hold said liquid in contact withan electrode in that barrel and connected to a circuit for controlling avoltage between said first and second electrodes during use; (b) an xyzcontroller attached to said nanopipette for effecting mechanicalmovement of the nanopipette in submicron x and y steps, and effectingmovement of said nanopipette in a z direction, said xyz controllerfurther having electronic controls for controlling said mechanicalmovements according to user defined control; and (c) said circuit forcontrolling a voltage connected to said electronic controls so that saidcircuit applies an ejection voltage to said liquid at a desired locationin said predefined location where liquid in said nanopipette is to bedeposited and removing said ejection voltage when the xyz controllereffects mechanical movement of the nanopipette away from the desiredlocation in said predetermined location where liquid is to be deposited.

In certain aspects of the invention, a substrate may have a uniformlayer on which the patterning material is deposited. The patterningmaterial can define an adhesion material for cell growth.

In certain aspects of the invention, the apparatus comprises a circuitfor controlling voltage that comprises a low noise amplifier for pipettebias and current measurement of ionic current through the first barrel.The apparatus may further comprise a piezoelectric actuator forsubmicron control of the xyz controller. The apparatus further maycomprise a programmable device, such as a computer or an FPGA for userinput of said predefined location. The FPGA may be programmed fordepositing spots in a pattern, so that cells grow in a predefinedpattern. The FPGA may be programmed to inject a cell at a predeterminedlocation. This location may be adapted to hold an individual cell inplace so that it can be scanned and injected. The predetermined locationmay be an organelle within a cell, such as a nucleus or a mitochondrion.

In certain aspects of the invention, the cell substrate is adapted tohold cells by having therein defined cavities for receiving anindividual cell in a single cavity. The cavity may have through-holesfor applying negative pressure to hold a cell in a cavity. The cavitymay comprise an electrode for attracting a cell to the cavity. Thecavity may serve to hold an individual cell in a fixed position while ananopipette is contacted with the cell, and also while it is injectedinto the cell.

Certain aspects of the invention comprise a method for depositing onto asubstrate a liquid in a predefined pattern having submicron features,comprising: (a) placing a liquid to be deposited into a multi-barrelednanopipette, wherein at least two barrels of said multibarreled pipettehave inside them a first and second electrode, said first electrode in abarrel adapted to hold said liquid in contact with an electrode in thatbarrel and connected to a circuit for controlling a voltage between saidfirst and second electrodes during use; (b) actuating an xyz controllerattached to said nanopipette for effecting mechanical movement of thenanopipette in submicron x and y steps, and effecting movement of saidnanopipette in a z direction, said xyz controller further havingelectronic controls for controlling said mechanical movements accordingto a user defined pattern, said actuating thereby moving the nanopipettein said predefined pattern; and (c) using said circuit for controlling avoltage connected to said electronic controls so that said circuitapplies an ejection voltage to said liquid at a desired location in saidpredefined pattern where liquid in said nanopipette is to be depositedand removing said ejection voltage when the xyz controller effectsmechanical movement of the nanopipette away from the desired location insaid predetermined pattern where liquid is to be deposited. Thesemethods may be further carried out according to specific embodiments ofthe apparatus defined above.

Certain aspects of the invention comprise a method for injecting amaterial into a cell on a substrate, using apparatus as described above.These methods may comprise the step of immobilizing said cell on saidsubstrate by placing said cell in a cavity in the substrate, said cavitysized to hold only an individual cell. Immobilizing may compriseapplying a voltage to the cell to hold it in place, or applying apressure differential across said cavity to aid in immobilizing saidcell. The cell may be injected with a variety of materials that are in abore of the nanopipette, including a polynucleic acid, an antibody, anda dye.

Certain aspects of the invention comprise an apparatus for measuring ananalyte within a single cell, comprising: (a) a nanopipette having abarrel containing a first electrode, said first electrode adapted tohold said analyte in contact with a second, reference electrode in aliquid contacting said cell and connected to a circuit for controlling avoltage between said first and second electrodes during use; (b) an xyzcontroller attached to said nanopipette for effecting mechanicalmovement of the nanopipette in submicron x and y steps, and effectingmovement of said nanopipette in a z direction, said xyz controllerfurther having electronic controls for controlling said mechanicalmovements according to user defined control; and (c) said nanopipettetip having an analyte binding material immobilized on an interiorsurface within said cell near a tip. This type of sensing an analytewith a functionalized nanopipette tip also involves inserting the tipinto or in the vicinity of an individual cell. The tip is functionalizedwith an analyte binding material such as a protein or a polynucleic acidintended to bind specifically to the analyte being detected or measured.Binding of the analyte affects current flow through the nanopore at thetip of the nanopipette. A protein for sensing the analyte may beimmobilized at the tip by sulfo-SMCC linked to a PLL coating on aninterior surface of the nanopipette. The nanopipette tip may befunctionalized with antibodies or aptamers, or receptors to a ligand tobe detected. The apparatus may be used in an immunoassay inside of asingle living cell.

Thus an apparatus of the present invention can be created in a number ofways for different applications. In each case, the device contains ananopipette attached to a sensitive xyz controller.

In certain aspects, the present invention comprises an apparatus formanipulating individual cells on a substrate, comprising: (a) amulti-barreled nanopipette, having (i) a first barrel containing a firstelectrode arranged to be in contact with a liquid in the first barrel;(ii) a second barrel adjacent the first barrel containing a secondelectrode arranged to be in contact with a liquid in the second barrel;and (iii) an amplifier connected to said first electrode for controllinga voltage between said first and second electrodes; (b) an xyzcontroller attached to said multi-barreled nanopipette for effectingmechanical movements of the multi-barreled nanopipette in submicron xand y steps, and effecting movement of said multi-barreled nanopipettein a z direction towards or away from the substrate, said xyz controllerfurther having electronic controls for controlling said mechanicalmovements according to user defined control; and (c) a circuit forcontrolling voltage between said first electrode and said secondelectrode and for applying an ejection voltage to said first electrode,said voltage sufficient to eject liquid from the first barrel at adesired location where liquid in said nanopipette is to be ejected andremoving said ejection voltage at least when the xyz controller effectsmechanical movement of the nanopipette away from the desired location.

In certain aspects, the present invention comprises an apparatus whereinthe detecting is detecting of specific oncoproteins in a single livingcell.

In certain aspects, the present invention comprises an apparatus whereinsaid circuit for controlling voltage comprises a low noise amplifier forproviding a pipette bias voltage and for and current measurement ofionic current through the first barrel.

In certain aspects, the present invention comprises an apparatus havinga piezoelectric actuator for submicron control of the xyz controller. Incertain aspects, the present invention comprises a field-programmablegate array (FPGA) for user input to determine said desired location. Incertain aspects, the present invention comprises an apparatus whereinsaid FPGA is programmed for depositing spots in a pattern on saidsubstrate. In certain aspects, the present invention comprises anapparatus wherein said FPGA is programmed to inject a cell which is at apredetermined location on said substrate. In certain aspects, thepresent invention comprises an apparatus wherein said FPGA is programmedto inject an organelle within said cell.

In certain aspects, the present invention comprises an apparatusoperatively connected to a substrate adapted to contain a plurality ofindividual cells, one cell each, in individual locations. In certainaspects, the present invention comprises a substrate that has cavitiesdefined therein for receiving an individual cell in a single cavity. Incertain aspects, the present invention comprises an apparatus whereinthe substrate comprises through-holes for applying negative pressure tohold a cell in a cavity. In certain aspects, the present inventioncomprises an apparatus wherein the cavity comprises an electrode forattracting a cell to the cavity.

In certain aspects, the present invention comprises a method fordepositing a liquid in a predefined pattern having submicron features,comprising the steps of: (a) placing a liquid to be deposited into amulti-barreled nanopipette having (i) a first barrel containing a firstelectrode arranged to be in contact with a liquid in the first barrel;(ii) a second barrel adjacent the first barrel containing a secondelectrode arranged to be in contact with a liquid in the second barrel;and (iii) an amplifier connected to said first electrode for controllinga voltage between said first and second electrodes; (b) actuating an xyzcontroller attached to said multi-barreled nanopipette for effectingmechanical movements of the multi-barreled nanopipette in submicron xand y steps, and effecting movement of said multi-barreled nanopipettein a z direction towards or away from the substrate, said xyz controllerfurther having electronic controls for controlling said mechanicalmovements according to user defined control; and (c) using a circuit forcontrolling voltage between said first electrode and said secondelectrode and for applying an ejection voltage to said first electrodeto eject liquid from the first barrel at a desired location where liquidin said nanopipette is to be ejected and removing said ejection voltagewhen the xyz controller effects mechanical movement of the nanopipetteaway from the desired location, whereby said liquid is deposited in saidpredetermined pattern.

In certain aspects the invention comprises a method as described abovewherein said liquid comprises a cell adhesion material for allowingattachment of cells on a substrate only at areas of deposited celladhesion material. In certain aspects the invention comprises a methodas described above wherein said cell adhesion material is laminin. Incertain aspects the invention comprises a method as described abovewherein the substrate comprises a uniform polymer top surface. Incertain aspects the invention comprises a method as described abovewherein said liquid comprises a cell adhesion material for allowingattachment of cells on the substrate only at areas of deposited celladhesion material, and further comprising the step of applying to thesubstrate adherent cells to be cultured, whereby said cells only adhereto regions having cell adhesion material.

In certain aspects the invention comprises a method as described abovewherein regions having cell adhesion material are at a density of aboutat least 10 spots per 100 square micrometers. In certain aspects theinvention comprises a method as described above wherein the method isused to deposit different material from different barrels of thenanopipette.

In certain aspects the invention comprises a method as described abovewherein the nanopipette is made of quartz and has an opening of about 20to 100 nm.

In certain aspects, the present invention comprises a method forinjecting a material into a selected cell on a substrate, comprising thesteps of: (a) placing a liquid to be injected into a multi-barrelednanopipette, having (i) a first barrel containing a first electrodearranged to be in contact with a liquid in the first barrel; (ii) asecond barrel adjacent the first barrel containing a second electrodearranged to be in contact with a liquid in the second barrel; and (iii)an amplifier connected to said first electrode for controlling a voltagebetween said first and second electrodes; (b) actuating an xyzcontroller attached to said multi-barreled nanopipette for effectingmechanical movements of the multi-barreled nanopipette in submicron xand y steps, and effecting movement of said multi-barreled nanopipettein a z direction towards or away from the substrate, said xyz controllerfurther having electronic controls for controlling said mechanicalmovements according to user defined control; and (c) using a circuit forcontrolling voltage between said first electrode and said secondelectrode and for applying an ejection voltage to said first electrodeto eject liquid from the first barrel at a desired location where liquidin said nanopipette is to be ejected and removing said ejection voltagewhen the xyz controller effects mechanical movement of the nanopipetteaway from the desired location, whereby said liquid is injected intosaid cell.

In certain aspects the invention comprises a method as described abovefurther comprising the step of immobilizing said cell on said substrateby placing said cell in a cavity in the substrate, said cavity sized tohold only an individual cell.

In certain aspects the invention comprises a method as described abovefurther comprising the step of applying a pressure differential acrosssaid cavity to aid in immobilizing said cell.

In certain aspects the invention comprises a method as described abovewherein said material injected is selected from the group consisting ofa polynucleic acid, an antibody, and a dye.

In certain aspects, the present invention comprises a apparatus fordetecting an analyte in an individual cell on a substrate, comprising:(a) a nanopipette having a barrel containing a first electrode, saidfirst electrode adapted to hold said analyte in contact with a second,reference electrode in a liquid contacting said cell and connected to acircuit for controlling a voltage between said first and secondelectrodes during use; and (b) an xyz controller attached to saidnanopipette for effecting mechanical movement of the nanopipette insubmicron x and y steps, and effecting movement of said nanopipette in az direction, said xyz controller further having electronic controls forcontrolling said mechanical movements according to user defined control;and (c) said nanopipette tip having an analyte binding materialimmobilized on an interior surface within said cell near a tip.

In certain aspects, the present invention comprises an apparatus whereinsaid analyte binding material is a protein or a polynucleic acid.

In certain aspects, the present invention comprises an apparatus whereinthe analyte binding material is a protein linked bysulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC) to a poly-L-lysine (PLL) coating on said interior surface.

In certain aspects, the present invention comprises a method to detectbiomolecules in a single living cell comprising: (a) positioning afunctionalized nanopipette to a defined depth into the cell; (b)monitoring the current to control precise position of nanopipette nearthe cell membrane; (c) inserting nanopipette at high speed into thecell; (d) applying high voltage; and (e) measuring current change todetect biomolecules in the cell wherein the functionalized nanopipetteis comprised in a system further comprising an amplifier for pipettebias and current measurement, a micromanipulator for control in the X, Yand Z directions, a piezo-actuator for fine control in the X, Y and Zdirections, a configurable integrated circuit, a high voltage source,and a relay for switching between low voltage and high voltage.

In certain aspects the invention comprises a method as described abovewherein the nanopipette is functionalized with antibodies. In certainaspects the invention comprises a method as described above wherein thenanopipette is functionalized with aptamers.

In certain aspects the invention comprises a method as described abovewherein the method is used in an immunoassay in a single living cell. Incertain aspects the invention comprises a method as described abovewherein the nanopipette is functionalized with receptor ligands.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the present of deposition and cellinjection system.

FIG. 2 is a schematic drawing of a cell penetration using adouble-barrel pipette with the present system.

FIG. 3 is a graph showing volume of liquid ejected from a nanopipettetip over time. As indicated in the graph, the ejection rate is linearover time.

FIG. 4A is a top view photograph that shows an array of patterned spots.

FIG. 4B is a pseudo-3-D graph of fluorescence image of an array ofpatterned spots using sulforhodamine.

FIG. 5 is a series of photographs showing the present nanoinjectiondevice. FIG. 5A is a photograph that show injection ofcarboxyfluorescein into HeLa cells. It is a brightfield image of cellsand nanopipette. Arrow indicates tip of nanopipette FIG. 5B is also aphotograph of cell injection, as in FIG. 5A. FIG. 5B shows a fluorescentimage of HeLa cell and nanopipette after injection.

FIG. 6 is a series of drawings that show cell sifter fabrication. FIG.6A is a drawing that shows silicon nitride layer is deposited on siliconwafer. FIG. 6B is a drawing that shows deep reactive ion etch throughnitride layer. FIG. 6C is a drawing that shows back side KOH etch. FIG.6D is a drawing that shows silicon wafer bonded to glass wafer. FIG. 6Eis a drawing that shows that, in the final device, a plastic ring isattached to surface.

FIG. 7 is a photograph of the final device showing a culture chamber anda fitting for applying a vacuum. A penny is shown for illustratingrelative size of the device as being smaller than a penny. Using thisdevice, cells can be cultured in the culture ring and immobilized onthrough holes by application of negative pressure.

FIG. 8 is a photograph that shows a fluorescence image of 10 mmpolystyrene beads immobilized on a cell sifter.

FIG. 9A is a cartoon depicting the topographical mapping with thescanning ion conductance microscopy (SICM). FIG. 9B is a graph of thetopographical mapping, showing height of the nanopipette tip changingrelative to location over a cell.

FIG. 10 is a line tracing showing selective detection of HPV18E6antigens by nanopipette sensors. Applied Voltage: −400 mV. Antigensadded at time 0.

FIG. 11 is a bar graph that shows the response of nanopipette sensors toHeLa cells lysate.

FIG. 12 is a bar graph that shows the detection of oncoproteins inside asingle cell with nanopipette sensors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview

Disclosed herein is a system comprising a nanopipette that is attachedto a robotic mechanism for three dimensional movement and useful forcontrolled deposition of material from the nanopipette onto a substratefor controlling cell growth, for sensing with the nanopipette tipbiochemical markers at individual cell surfaces or cell interiors, orfor injection of nanopipette contents into single cells. When used forchemical deposition, the small size of the nanopipette opening allowsfor pattern definition with resolution below the single cell level. Thesystem further comprises using scanning ion conductance microscopy(SICM) as a basis for pattern deposition. Thus, a pattern can bepredefined by the user, and subsequently laid down by controllingsoftware, without the need for photolithography or any type of patternpreprocessing. This allows for the constant, rapid development ofarbitrary user defined patterns to be quickly and efficiently evaluatedwith in vitro cell cultures and no loss of resolution. The disclosedsystem has applications in biological studies for controlling cellularattachment and growth.

SICM devices have been previously devised for imaging the topography ofsoft non-conducting surfaces. See, e.g. Hansma, P. K., et al., TheScanning Ion-Conductance Microscope. Science, 1989. 243(4891): p.641-643. Such prior art devices use XYZ scanning, Z feedback and controllogic (Z is considered to be the direction orthogonal to the surfacebeing scanned).

Another aspect of the present invention is directed to a systemcomprising a nanopipette for high-throughput cell injection includingmethods for cell immobilization and injection. Embodiments of theinvention are directed to cell injection of an arbitrary material into acell using a nanopipette and electroosmotic injection, with minimaldamage to the cell. Electroosmotic injection involves changing a voltageand ionic current across a nanopore opening to drive a compound ofinterest out of the nanopipette. Also disclosed are double-barrelnanopipettes for higher efficacy of ejection and cell injection.

Also disclosed is a cell sifter fabricated for immobilization of cellsin a predetermined array for fully automated injection into cells. Thecell sifter contains three dimensional features, such as recessedopening, that allow for the placement of single cells that may beretained in a predetermined location, preferably in an array oflocations, each containing a single cell. Embodiments of this technologycan be used as a tool for high throughput single-cell studies involvingthe introduction of material reliably into a single living cell orimmunoassays of the cell interior. Alternate embodiments includeimmobilization of structures including polymer beads.

Also disclosed are methods and devices integrating the presentnanopipette sensors with apparatus for scanning the nanopipette across abiological material. A nanopipette sensor as described here can becoupled with scanning ion conductance microscopy (SICM) to studymolecular interactions inside individual living cells. Cells can beinterrogated inside the growing medium without any need for fixation orpretreatments. The small size of the nanopipette with respect to thecell size combined with controlling the conditions of penetrationmaximizes cell viability. Applications of this technology include invivo assay down to the single cell level. Another application is itallows for the continuous monitoring of biomolecules inside individualcells, e.g., an immunoassay to monitor protein expression. Furthermore,this technology can be employed to functional map secreted moleculesfrom a single cell.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes of theclarity, following terms are defined below.

The term “nanopipette” is used herein to refer to a hollowself-supporting, inert, non-biological structure with a conical tipopening (i.e. a nanopore) of nanoscale, i.e., 0.05 nm to about 500 nm,preferably about (+ or −20%) 50 nm. The hollow structure may be glass orquartz, and is suitable for holding inside of it, or on one side of thenanopore, a fluid which is passed through the nanopore opening. Theinterior of the nanopipette is selected or modified to minimizenonspecific binding of analyte. The interior is sized to allow insertionof an electrode that contacts solution in the nanopipette. Nanopipettesare preferably fabricated by laser pulling of glass or quartz capillarytubes. Nanopipettes have an inner diameter on the order of 10-100 nm, anouter diameter on the order of 200-800 nm, and a typical length of about10 μm. These dimensions are sized to suit a particular application, andcontrol of the nanopore size is an important consideration. A“multibarrelled nanopipette” is a nanopipette that has two or moreparallel bores that typically share a common wall. The bores areco-axial, typically radially spaced, but may be concentric. They may befabricated from multibore capillary tubes, which are commerciallyavailable.

The term “nanopore” is used herein to refer to a small hole in anelectrically insulating membrane that can be used as a single-moleculedetector. The detection principle is based on monitoring the ioniccurrent passing through the nanopore as a voltage is applied across themembrane. A preferred nanopore opening is between about (within 10% of)20 and 100 nm.

The term “current detecting circuit” or “circuit for controlling avoltage” refers to known electronic circuits and devices that include acontrollable amplifier and a sensitive voltage and current detector.They may comprise any sensitive device for detecting changes in currenton the order of 1-10 picoamperes, based on a baseline current of10-10000 picoamperes. The term further refers to a circuit that is timeresponsive and relatively temperature independent or allow for changesin temperature to be compensated for. It should have an input in acircuit where a known voltage is supplied. Sensitive detecting circuitsare known, including voltage clamp amplifiers and transimpedanceamplifiers. The term “voltage clamp” here refers to circuits whichutilize a differential amplifier having one input connected to avariable command voltage, another input connected to a measured voltage,and a feedback circuit. The voltage clamp uses negative feedback tomaintain the system at the command voltage, which in this case is apredetermined alternating signal, such as an alternating voltage signalfrom a signal generator. The output current follows changes in the inputvoltage and small changes in current can be detected.

The term “electroosmotic flow” or “electro-osmotic flow” is used hereinin its conventional sense to refer to the effect of an applied electricfield on the electrical double layer that forms at the surface of thenanopipette. The ions arranged at the inner surface of the nanopipettetip are forced out of the nanopipette under a sufficiently strongelectric field, such that a small volume of solution is simultaneouslyforced from the tip. Electroosmosis ejects a liquid, whileelectrophoresis moves charged particles within a medium such as liquidor gel. Electrosomosis is induced by an applied potential across acapillary tube, or any other fluid conduit.

The term “laminin” is used herein in its conventional sense to refer tomajor proteins in the basal lamina (formerly improperly called “basementmembrane”), a protein network foundation for most cells and organs. Thelaminins are an important and biologically active part of the basallamina, influencing cell differentiation, migration, adhesion as well asphenotype and survival. Laminins are trimeric proteins that contain anα-chain, a β-chain, and a γ-chain, found in five, three, and threegenetic variants, respectively. Laminin is vital for the maintenance andsurvival of tissues. Defective laminins can cause muscles to formimproperly, leading to a form of muscular dystrophy, lethal skinblistering disease (junctional epidermolysis bullosa) and defects of thekidney filter (nephrotic syndrome). Laminin is used in cell culture andfor studying cellular interactions with the extracellular environment.For example, Laminin-111 is a major substrate along which nerve axonswill grow, both in vivo and in vitro. For example, it lays down a paththat developing retinal ganglion cells follow on their way from theretina to the tectum. It is also often used as a substrate in cellculture experiments.

The term “oncoprotein” is used herein in its conventional sense to referto a protein encoded by an oncogene; they may include viral proteinsknown to be associated with cancer. See, e.g. Clemens et al.,“Dimerization of Human Papillomavirus E7 Oncoprotien in Vivo,’ Virology214, 289-293 (1995). The protein encoded by the viral oncogene and thecorresponding, homologous protein within the host cell are both referredto herein as oncoproteins, although the cellular oncoprotein istypically larger and is present in small quantities in normal cells, andthus need not only be associated with neo-plastic states. They includemyc, bcl-2, mutated p53, DEK, HPV E6, HPV E7, etc.

The term “cell adhesion material” is used herein to refer to materialssuch as proteins that for example, in nature, attach cells to specificcompounds in the extracellular matrix (a process known as celladhesion). Some of the amino acids in the substrate adhesion molecules(SAM) bind to components of the extracellular matrix, while others bindto integrins on the surface of the cell. Integrin molecules are composedof two chains of amino acids, one of which is connected to the actinfilaments in the cytoskeleton, while the other is connected to the SAMs.This enables external activity in the extracellular matrix to affect theshape and movement of the cell. SAMs do not have to be made by the cellsthat bind to them. They can also link to other SAMs, influencing eachother's behavior.

The term “field-programmable gate array” is used herein to refer to anintegrated circuit designed to be configured by the customer or designerafter manufacturing—hence “field-programmable”. The field-programmablegate array (FPGA) configuration is generally specified using a hardwaredescription language (HDL), similar to that used for anapplication-specific integrated circuit (ASIC). FPGAs containprogrammable logic components called “logic blocks”, and a hierarchy ofreconfigurable interconnects that allow the blocks to be “wiredtogether”—somewhat like many (changeable) logic gates that can beinter-wired in (many) different configurations. Logic blocks can beconfigured to perform complex combinational functions, or merely simplelogic gates like AND and XOR. In most FPGAs, the logic blocks alsoinclude memory elements, which may be simple flip-flops or more completeblocks of memory.

The term “xyz controller” refers to a mechanical device that moves inthree dimensions, known as the x and y dimension, typically a flatsurface, and the z direction, typically vertical. Xyz controllers areknown for use in a variety of micro-scale applications, such as anatomic probe microscope; see, e.g. U.S. Pat. No. 5,394,741 to Kajimuraet al., entitled “Atomic probe microscope,” for further details on anexemplary xyz controller.

The term “ejection voltage,” as further described below, refers to avoltage applied in the present nanopipette between the bore, containinga solution, and an exterior solution, which voltage causes an ionic flowfrom the bore, i.e. ejecting an atomic species within the solution. Thatis, in the present device, ejection from the nanopipette bore occurs dueto electroosmosis, which means it depends on the properties of thenanopipette rather than what is being ejected. The fluid is ejected fromthe bore that contains a positively biased electrode, relative to thecounter electrode that is in the other bore. As exemplified below, avoltage between 0.1 to 100V is applied across the two bores of a doublebarreled nanopipette to cause ejection. The amount of ejected materialdepends on the time and the magnitude of the applied voltage. Forpurposes of illustration, low voltage is between 0.01-1V and highvoltage is 1-100V. If a single bore pipette is used and a high voltagerelative to an electrode outside the cell is applied, then the voltagewould have to pass through the cell membrane likely causing damage.Thus, a double bore pipette is preferred over the use of a single borepipette.

The term “low noise amplifier,” or LNA as is known in the art, refers toone of a variety of amplifiers, where the amplifier is an electronicamplifier used to amplify very weak signals (for example, captured by anantenna). It is usually located very close to the detection device toreduce losses in the feedline. Typically, using an LNA, the effect ofnoise from subsequent stages of the receive chain is reduced by the gainof the LNA, while the noise of the LNA itself is injected directly intothe received signal. Thus, it is necessary for an LNA to boost thedesired signal power while adding as little noise and distortion aspossible, so that the retrieval of this signal is possible in the laterstages in the system. A good LNA has a low NF (like 1 dB), a largeenough gain (like 20 dB) and should have large enough intermodulationand compression point (IP3 and P1dB).

The term “piezo actuator,” as is known in the art, refers to apiezoelectric transducer. The active element is basically a piece ofpolarized material (i.e. some parts of the molecule are positivelycharged, while other parts of the molecule are negatively charged) withelectrodes attached to two of its opposite faces. When an electric fieldis applied across the material, the polarized molecules will alignthemselves with the electric field, resulting in induced dipoles withinthe molecular or crystal structure of the material. This alignment ofmolecules will cause the material to change dimensions. In terms of thepresent device, the transducer is used to accomplish fine degrees ofmovement on a very small scale.

The term “polynucleic acid” refers to any oligomer or polymer of singleor (partially) double stranded DNA or RNA or synthetic analogs thereof;as used herein, the polynucleic acid is capable of binding to acomplementary strand with specificity based on Watson-Crick basepairing.

The term “antibody” refers to any antibody or antibody fragments havingbinding specificity through an antigen binding portion. For example, ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR).

The term “aptamer” refers to a polynucleotide or peptide that binds to aspecific target molecule. Exemplary aptamers include those derived fromSELEX protocols, as described e.g. in Gold et al. U.S. Pat. No.5,567,588 entitled “Systematic evolution of ligands by exponentialenrichment: Solution SELEX;” SELEX-identified oligonucleotidescontaining modified nucleotides are described, e.g., in U.S. Pat. No.5,660,985, which describes oligonucleotides containing nucleotidederivatives chemically modified at the 2′ position of ribose, 5 positionof pyrimidines and 8 position of purines, U.S. Pat. No. 5,756,703 whichdescribes oligonucleotides containing various 2′-modified pyrimidinesand U.S. Pat. No. 5,580,737 which describes highly specificoligonucleotides containing one or more nucleotides modified with2′-amino (2′—NH2), 2′-fluoro (2′-F) and/or 2′-O-methyl (2′-OMe)substituents. Peptide aptamers are combinatorial recognition proteins.Peptide aptamers have been selected, using yeast two-hybrid methods, tobind to a wide range of cellular, viral and bacterial target proteinsinvolved in a variety of regulatory pathways, as described in Colas, Theeleven-year switch of peptide aptamers,” J. Biol. 7(1) 2 (2008). Apeptide aptamer is described in US 20090318363 entitled “Peptide aptamerfor neutralizing the binding of platelet antigene specific antibodiesand diagnostic and therapeutic applications containing the same.”

The term “dye” refers to a composition that imparts color to anothercomposition of matter or to a mixture. Preferred dyes are fluorogeniccompounds. Also included in this definition are energy transfer dyes.Also included are xanthene dyes, asymmetric benzoxanthene dyes,rhodamine dyes, fluorescein dyes, 4,7-dichlororhodamine dyes,4,7-dichlorofluorescein dyes, carboxyfluorescein dyes, carboxyrhodaminedyes, carboxy R110 dyes, carboxy R6G dyes, carboxy-X-rhodamine dyes,cyanine dyes, phthalocyanine dyes, squaraine dyes, and Cy5, as definedor used in U.S. Pat. No. 6,335,440, which is also hereby incorporated byreference.

General Method and Apparatus

In a first aspect, the present invention comprises a system fordepositing a user defined pattern on an arbitrary substrate for thepurpose of controlled cell adhesion and growth. The approach has theadvantage that controlled deposition can be performed onto anunfunctionalized surface in an ambient environment. Such patternedsubstrates are used for successful cell cultivation and good attachmentand viability to predefined patterns.

Device as Adapted for Substrate Patterning

The system comprises a computer controlled feedback system exemplifiedby a system for scanning ion conductance microscopy (SICM). The basicoperation of SICMs is well understood in art (See, for example, Hansma,P. K., et al., The Scanning Ion-Conductance Microscope. Science, 1989.243(4891): p. 641-643; Prater, C. B., et al., Scanning Ion-ConductanceMicroscope And Atomic Force Microscope. Scanning, 1990. 12(1): p.50-52). Briefly, the basic SICM consists of a single pipette back-filledwith an electrolyte and immersed in an electrolytic bath. An electrodeis placed in the nanopipette and a ground electrode is placed at somedistance away in the bath. As the opening of a nanopipette approaches asubstrate, normal to the surface, the current flowing through thepipette orifice for a given will be reduced by “current squeezing”. Thisdrop in current can act as a feedback signal to control the height ofthe nanopipette which can be controlled at a predefined distance abovethe surface. If the nanopipette is then scanned across a surface atopographical image can be rendered of the substrate.

As explained below, features of this device may be incorporated intorelated devices for injecting a single cell, or for measuring molecularbinding within a single cell.

FIG. 1 is a schematic drawing of the present device, which can beincluded in a modified SICM (scanning ion conductance microscopy) custombuilt for cell injection according to the present invention. The presentdevice relies on feedback-controlled positioning in the z-direction forfine control over penetration of the membrane. According to the presentdevice, a double-barrel nanopipette 102 is attached to the xyzcontrolling portion, comprising a piezo x,y,z controller 104 and a piezocontroller 106 operatively connected to the piezo x,y,z controller. Thepiezo xyz controller 104 is used for very fine movement, and is mountedon a step-motor micromanipulator (not shown) for more coarse movement. Apiezo control circuit is connected through a DAQ card 114 to a computer116. The DAQ is connected to an electronic sensor and amplifierconnected to an electrode 110 within the nanopipette. (A referenceelectrode 122 in the second bore, fused to the first bore, is connectedto ground, as shown.) The SICM module further comprises a low-noiseamplifier 108 (for example, Axopatch 200B, Molecular Devices, Sunnyvale,Calif.) for pipette bias and current measurement. The amplifier isconnected through DAQ card 114 to an electrode 110 within thenanopipette, as shown. It includes a headstage amplifier 112 feedinginto the amplifier 108, which outputs to a DAQ (data acquisition) card114. Suitable DAQ cards are commercially available. For example, DAQboards (made by National Instruments) are multi function plug-n-play,analog and digital input/output boards consisting of a onboard timer, 12bit analog to digital converter (ADC) with 8 channel input, 2 digital toanalog converters (DAC) and 24 TTL level logic inputs.

The DAQ card (which is a FPGA) interfaced with a computer controller116, also is connected to the circuitry for controlling movement of thenanopipette through a piezo xyz controller. One xyz controller is amicromanipulator 106 (for example, MP-285) for coarse control in the X,Y, and Z directions. This is then coupled to a piezo actuator 104 forfine control (positionable within 2-3 nm) in the X, Y, and Z directions,and a FPGA for hardware control of the system. The system is furthermodified with a low-noise high voltage source 118 (for example, Model2100 Isolated Pulse Stimulator, A-M Systems), and a custom relay 120 forswitching between the low voltages (about 0.01-1 V) required forfeedback control and the high voltages needed for electrophoreticmaterial ejection (FIG. 2). The system is controlled using user codedsoftware written, for example, in LabVIEW. LabVIEW is available fromNational Instruments Corporation, and LabVIEW is a graphical programmingenvironment used to develop sophisticated measurement, test, and controlsystems using intuitive graphical icons and wires that resemble aflowchart. Thus a user can design a predetermined pattern of nanopipettexyz motion, as well as voltage changes to cause ejection of material ata predetermined location, for a predetermined length of time. LabViewalso permits a predetermined pattern to be loaded into the software.LabView or other conventional software is supplied with the presentdevice to control the sensing and ejection from the nanopipette, and themotion of the nanopipette.

Material ejection from a bore of the pipette is performed using adouble-barrel nanopipette back filled with an appropriate buffer and thematerial of interest. Suitable buffers include phosphate, citrate or anyother buffers routinely used in molecular studies. An ejection voltageis applied between electrodes 110 and 122 by changing the voltage comingfrom the power supply 118.

A variety of electrolyte solutions may be used in the nanopipette boreto affect ejection of the material of interest. In the case of cellpatterning, the material of interest will be used to create a celladhesion pattern. As described below, the material of interest may bevaried, depending on the application. The material of interest issuspended in the bore in an electrolyte solution, which, as is known,contains dissolved electrolytes, i.e., free ions. Typical ions includesodium, potassium, calcium, magnesium, chloride, phosphate andbicarbonate. Other ionic species may be used. The material of interestwill typically be liquid, in that it will comprise the material ofinterest, and the ions in solution. The material of interest itself maybe an electrolyte, such as human plasma or other body fluids, solutions,water samples and so on. The electrolyte should carry an ionic current;about 10-100 mM, preferably about 100 mM of positive and negative ionicspecies are thought to be required for this function. The present devicemay employ either the same or different electrolytes in the nanopipetteinterior and in the sample material. A variety of salts may be used inthe electrolyte solution. They are composed of cations (positivelycharged ions) and anions (negative ions) so that the product iselectrically neutral (without a net charge). These component ions can beinorganic such as chloride (Cl⁻), as well as organic such as acetate(CH₃COO⁻) and monatomic ions such as fluoride (F⁻), as well aspolyatomic ions such as sulfate (SO₄ ²⁻). There are several varieties ofsalts. Salts that hydrolyze to produce hydroxide ions when dissolved inwater are basic salts and salts that hydrolyze to produce hydronium ionsin water are acid salts. Neutral salts are those that are neither acidnor basic salts. Molten salts and solutions containing dissolved salts(e.g. sodium chloride in water) are called electrolytes, as they areable to conduct electricity.

Material of interest to be ejected includes, but is not limited to,small molecules, metabolites, nucleic acids, oligonucleotides, peptides,amino acids, dyes, polymers, nanoparticles

Nanopipettes may be fabricated using quartz. Double-barrel pipettesoffer the advantage of forming a small meniscus across the tip of thepipette negating the need for an external ground electrode as is commonwith single-barrel pipettes. Additionally, because the circuit iscompleted between the two barrels of the pipette the tip can function asa SICM in air, thus allowing the tip of the nanopipette to approach adry surface. It has been previously shown by Rodolfa et al in 2005(“Two-component graded deposition of biomolecules with a double-barrelednanopipette.” in Angewandte Chemie-International Edition, 44: p.6854-6859) that material can be ejected from the tip of a nanopipetteusing electroosmotic forces and relatively high voltages. Thenanopipette is preferably formed of fused silica or amorphous quartz,which is less expensive than crystalline quartz. Crystalline quartz may,however, be utilized. Ceramics and glass ceramics and borosilicateglasses may also be utilized but accuracy is not as good as quartz. Theterm “quartz” is intended and defined to encompass that special materialas well as applicable ceramics, glass ceramics or borosilicate glasses.It should be noted that various types of glass or quartz may be used inthe present nanopipette fabrication. A primary consideration is theability of the material to be drawn to a narrow diameter opening.Alternatively, nanopipettes may be fabricated from other non-conductivematerial such as other silicon oxides, metal oxides, (e.g. aluminum),carbon (See, e.g. U.S. Pat. No. 7,597,941, Tubular carbon nano/microstructures and method of making same), etc.

In some embodiments, the system is used for cell patterning on asubstrate. Patterning is controlled using software programmed to controlnanopipette height and position. A predefined pattern is loaded into thesoftware. At the prescribed areas, the nanopipette is slowly lowered tothe surface of the substrate to be patterned. Once the meniscus of thepipette is in contact with the substrate the relay isolates theamplifier, and applies high voltage for a prescribed amount of time.After the elapsed time, the tip is quickly retracted, the voltage acrossthe pipette tip is discharged through a resistor to ground, and theamplifier is reconnected to the pipette. The tip is then moved to thenext point and the process is repeated until an entire pattern isdeposited. The rate of ejection is dependent both on applied voltage andon the duration of the applied voltage (see volume ejected versus timegraph in FIG. 3).

Using the presently described apparatus, volumes as low as between 50pL/s to 100 pL/s may be ejected from the nanopipette. Volumes can beeven lower than these amounts, but they would be beyond the resolutionof optical microscopes. Ejection volumes were determined by ejectingonto a suitable polymer surface in a bath of oil, for example mineraloil. Suitable liquids for contact between the tip and the substrateinclude organic liquid polymers (e.g. oil) and silicon-based polymers,e.g. silicones. An example of silicone is polydimethylsiloxane. Ahemispherical shape of the oil drop was assumed for the volumecalculations. Ejection volumes appear to be linear with the time ofapplied voltage. Spot sizes as small as 3 μm in diameter may bedeposited. FIG. 4 shows an example of an array of patterned spots ofsulforhodamine wherein the mean spot diameter is 4±0.8 μm. Resultspresented here are representative of individual nanopipettes; theparameters are obtained through linear fits to these data and can varybetween nanopipettes. Nevertheless for standardized array printing, eachnanopipette can be easily calibrated by ejecting droplet in oil.Additionally, by reversing the bias across the barrels, differentmaterial can be controllably ejected from different barrels. By loadingeach barrel with a separate material a multilayer pattern can be laiddown in a pre-registered fashion, in a single patterning run.

Patterns on the substrate where cells are to be attached for culture maybe defined on the substrate by using proteins, polymers includingorganic polymers, or biopolymers. Patterning is preferably used todefine a number of discrete areas where separate cell growth can takeplace. Examples of patterning materials include, but are not limited topolylysine, bovine berum albumin (BSA), and surface adhesion molecules,e.g. fibronectin or laminin. Pattern deposition may be confirmed bymixing quantum dots with the electrolyte solution. Patterns may beassessed using, for example, fluorescence microscopy to visualizedeposition, and check for errors in printing. The substrate, upondeposition, is then washed in a self masking step. Suitable buffers withmasking molecules may be used for the washing step. Examples ofmolecules used for masking include bovine serum albumin (BSA). Afterpattern definition the substrate may be immersed in cell culture media.Cells may be dispersed in media and allowed to settle onto thesubstrate. After several hours of incubation cells are gently rinsedwith pre-warmed culture media to remove any cells not firmly attached tothe substrate. Cell attachment is assessed both qualitatively andquantitatively.

Cell Injection

In another embodiment, the present nanopipette system is used forelectro-osmotic injection of material into a single living cell in ahigh-throughput manner and with minimal damage to the cell. An advantageof the approach is the highly automated nature of the injection, precisecontrol of injection volumes, and the high cell viability. Further, acell sifting device is presented below for cell immobilizationincreasing the potential for full system automation.

Cell injection may be performed using a double-barrel nanopipette (shownin FIG. 2) back filled with a suitable buffer and material to beinjected. Suitable buffers include phosphate, citrate or any otherbuffers routinely used in molecular biology studies. Material to beinjected includes, but is not limited to, small molecules, metabolites,nucleic acids, oligonucleotides, peptides, amino acids. FIG. 2illustrates cell penetration using a double-barrel pipette 202, havingbarrels 204, 206 and electrodes 208, 210 in the barrels, one in eachbarrel. The pipette 202 will be attached to a piezoelectric controlleras shown in FIG. 1. Double-barrel pipettes offer the advantage ofallowing one barrel to be biased relative to the other barrel which canact as a ground electrode (see electrode 122 in FIG. 1) negating theneed for an external ground electrode as is common with single-barrelpipettes. This has the further advantage of eliminating anyelectroporative effects which may occur with a single-barrel pipette andexternal ground electrode. The rate of ejection from the tip of ananopipette is dependent both on applied voltage and on the duration ofthe applied voltage (FIG. 3). Ejection volumes appear to be linear withthe time of applied voltage, which was 100V, with the pipette tipimmersed in mineral oil. Mineral oil was used solely for calibrationpurposes by measuring the droplet size of ejected material. The mineraloil contains the ejected material since aqueous solutions are notsoluble in mineral oil.

Injection of material into cell 212 from barrel 202 connected to thepower supply 118 is performed using the circuit described above in FIG.1 and controlled using custom software. Injection is shown as takingplace in the cytoplasmic region of cell 212, but injection into anobserved nucleus 214 could also be accomplished. The cell rests on asolid support 216, which may be created as shown in FIG. 6. Cellsincluding bacterial, fungal, animal, mammalian cells or cells from celllines may be used for injection purposes. Injection is accomplished byslowly lowering the nanopipette to within between 50 to 200 nm,preferably ˜100 nm, of the cell membrane where it enters feedback and isstopped. The nanopipette is then quickly lowered between 0.5 to 2 μm,preferably between 0.9-1.2 μm penetrating the cell membrane. The speedof the nanopipette while lowering may be between 50 to 200 μm/s,preferably more than 80 μm/s. The low-voltage feedback system isdisconnected from the nanopipette and a high-voltage source is connectedby high-voltage relay for a predetermined amount of time. The lowvoltages for the feedback include voltages less than 2V, preferably lessthan 1V. The high voltages for the feedback include voltages more than0.5V, preferably more than 1V. Two different power supplies areutilized; one for positioning the pipette that has a power supplyranging from −1 to +1V and another for injecting that ranges from −100Vto +100V. Typical cell injections range from about 5-30V, but these canbe varied as long as the ejection is reproducible and the voltage doesnot damage the cell. The nanopipette is then discharged through aresistor and the nanopipette is retracted at high-speed. FIG. 5 shows anexample of injection of a dye carboxyfluorescein into a HeLa cell. Nocell damage is immediately visualized, and confinement of fluorescenceis confined within the cell walls for several minutes (FIG. 5).

Cell Substrate Patterning

Another aspect of the present invention is directed to a cell sifterdesigned and fabricated for the immobilization of cells or molecularstructures including beads in an ordered array. By immobilizing cells inan ordered array, the SICM can be controlled to address each element ofthe array sequentially, reducing the need for an operator toindividually inject each cell separately. The sifter is fabricated usingstandard semiconductor fabrication technology (FIG. 6A-E). Briefly, a500 to 1000 nm layer, preferably ˜750 nm layer of a silicon salt (forexample, silicon nitride) 602 is deposited onto the surface of a siliconwafer 604. The silicon wafer may be between 200 to 1000 μm thick,preferably between 300-600 μm thick. 1 to 5 μm, preferably 2-3 μm holes606 are etched through the silicon salt layer in an array with asuitable pitch using a reactive ion etch. A suitable pitch is more than50 μm, preferably around 100 μm pitch. Fiduciary marks (not shown) areetched into one corner of the array which can be used for SICMalignment. The wafer backside 608 is then etched by a suitable alkali(for example, sodium hydroxide or potassium hydroxide) to define achamber 610 underneath the holes, as well as a channel 612 for theapplication of negative pressure to the chamber. The silicon wafer isthen bonded to a glass wafer 614 sealing both the etched chamber andchannel. The device is designed symmetrically so that two arrays arepresent at opposite ends of the device. The device is completed bybreaking out the silicon salt layer on one array and attaching a barb616 on which to attach a vacuum line. Around the other array a plasticring 618 is attached with a suitable polymer to act as a cell culturechamber allowing cells to be immersed in cell culture media. Suitablepolymers include organic polymers including silicon polymers e.g.,silicones. An example of a silicone is polydimethylsiloxane. The devicecan be used to immobilize molecules including, but not limited to,polymers. Molecules may be comprised in beads 620. The cells ormolecules are immobilized by applying negative pressure to the devicethrough-holes. The cells would be placed in an array defined by theholes, as shown by exemplary beads 620

As an example, the device was tested using 10 μm fluorescent polystyrenebeads (yellow green-absorption: 505; emission: 515). The beads areroughly the size of mammalian cells and provide a good model for testingthe device. Beads were successfully immobilized by application ofnegative pressure to the device through-holes. After gentle washing itwas observed that all through-holes were immobilizing a singlepolystyrene bead over each hole (FIG. 6E). In another example, the cellsifter was used to immobilize HeLa cells. Cells were suspended in 0.1MPBS and successfully immobilized by application of negative pressure tothe through-holes.

Molecular Sensing within a Single Cell

Another aspect of the present invention is directed to the combinationof a nanopipette that is functionalized to act as a sensor and coupledwith an xyz controlling injection device, as shown in FIG. 1. Thenanopipette sensors are geometrically extremely similar to taperedoptical fiber; they can be fabricated with the same laser pullingprocess however the transduction mechanism is fundamentally different.Biodetection on optical fiber relies either on the detection offluorescently tagged analyte or on an ELISA-like read out whilenanopipette sensor readout is purely electrical and no biomoleculelabeling is needed. The sensitivity of the nanopipette sensor ismaximized at the conically shaped nanopipette tip, making the dimensionand geometry of the tip orifice crucial for biosensor performance.Gating, from permanent binding at the nanoscale-sized tip opening,causes distinctive changes to the nanopipette electrical signature. Theelectrical changes are then detected with a simple electrochemicalsetup, in real time, without any need for labeling. Like optical fibers,nanopipette sensors can be integrated with piezo actuators achievinghigh spatial resolution as it is exploited in Scanning Ion ConductanceMicroscopy. This combination of nanopipette sensors and SICM is used fortopographical mapping of surfaces including multiple and single cells.Another application is in detection of metabolites and moleculesincluding proteins and nucleic acids in single living cells usinglabel-free biosensor. In some embodiments, immunoassays can be performedinside a single living cell. Another application is in detection ofoncoproteins in single cancer cells. Another application is in themeasurement of bioactivity in single cells, in real time and withnanoscale resolution. For example, it can be used in studyingprotein-protein interactions in a single cell.

FIG. 9A is a cartoon depicting the topographical mapping of a cell usingthe technique embodied herein. The detection of molecules inside a cell902 relies on a high precision nanomanipulator and a current feedbackloop to insert the nanopipette sensor 904 to a defined depth into theprobe cell. As a nanopipette 904 approaches the surface of a cell, theionic current through the pipette 904, having a single bore and anelectrode 906 therein, will decrease due to “current squeezing,” a wellknown effect, exploited in scanning ion conductance microscopy. Bymonitoring the ionic current, the precise position of the nanopipettecan be determined and controlled within ˜200 nm of the cell membraneusing a feedback circuit and an xyz controller as shown in FIG. 1.Additionally, the feedback mechanism ensures that the nanopipette tipdoes not come into contact with anything but the cell membrane,preventing the tip from potentially breaking by coming into contact withother structures or substrates. Cells may be plated on an even surface908 (for example, a petri dish, glass slide) which is coated with alayer of a suitable polymer. Suitable polymers include organic polymersincluding silicon polymers e.g., silicones. An example of a silicone ispolydimethylsiloxane. Cells may be covered with a suitable bufferincluding, but not limited to, phosphate, citrate buffers. Thenanopipette sensor may be biased at a positive voltage (for examplevoltage between +100 mV to +1000 mV, preferably more than +500 mV) whenthe electroosmotic flow is directed outwards from the nanopipettebarrel. This configuration avoids any cellular debris or moleculespresent in the medium to interact with the sensor and affect itsperformance. The feedback loop lowers the sensor with steps of about 10nm until the current is reduced to 70 to 99%, preferably more than 99%of its initial value. After this initial positioning above the cellmembrane the nanopipette is inserted into the cell by about 2 μm,preferably more than 1 μm, at a speed of between 50 to 200 μm/s,preferably between 80-150 μm/s. The sensor is inserted at a very highspeed to reduce disturbance to the cell and maximize cell viability.Multiple penetrations can be performed on the same cell, without anynoticeable change in the nanopipette current baseline and viability ofthe cell. Once inserted inside the cell, an AC voltage with amplitude ofbetween 250 mV to 500 mV and frequency of between 1 to 10 Hz, preferablymore than 4 Hz is applied to the nanopipette sensor to perform thesensing.

The present nanopipette sensors comprise functionalized nanopipettes,used for the sensing component of the present devices. Functionalizednanopipettes are described in an earlier US patent applicationpublication by two of the inventors (US 2010/0072080, published Mar. 25,2010, titled “Functionalized Nanopipette Biosensor”) which isincorporated herein by reference. Briefly, nanopipettes may befunctionalized by chemical linkage to molecules including polymers,polysaccharides, and biomolecules including but not limited toantibodies, peptides and biopolymers. The sensors may be coated on aninterior surface with polyacrylic acid (PAA), a polymer of acrylic acidunits. The formula of PAA is (C₃H₄O₂)_(n). In a water solution atneutral pH, many of the side chains of PAA lose their protons andacquire a negative charge. This makes PAA a polyelectrolyte. The surfacemay be further functionalized to be bound to a polysaccharide or aprotein. Detection of binding to the nanopipette sensor is based oncurrent rectification. This refers to an effect when charged nanoporesrespond to a symmetric input voltage with an asymmetric current output.When the diffuse electrical double layer thickness is comparable withthe pore size, the electrostatic interactions between fixed chargedspecies on the nanopore surface and ionic species in solutions altersnanopipette permselectivity. The rectification coefficient, r, isdefined as the logarithm of the ratio between the current measured atparticular positive voltage and the current measured at the same voltagebut with the opposed polarity.

$r = {\log_{10}\frac{I_{+}}{I_{-}}}$

This coefficient is a useful indicator of the rectifying properties of ananopipette and therefore of the fixed charges on the sensor surface.Quartz nanopores, being negatively charged, show a negative currentrectification (r<0). The rectification can be inverted (r>0) bymodifying the nanopore surface with charged functional layers such aspoly-L-lysine, dendrimers, aminosilane and chitosan.

EXAMPLES Example 1 Materials and Methods

Double-Barrel Nanopipette Fabrication:

Nanopipettes were fabricated from theta quartz capillaries with an outerdiameter of 1.2 mm and an inner diameter of 0.90 mm (QT120-90-7.5;Sutter Instrument Co.). The capillary was then pulled using a P-2000laser puller (Sutter Instrument Co.) programmed with a two-line programto fabricate nanopipettes with an inner diameter of ˜50 nm. Parametersused were: Heat 650, Fil 4, Vel 20, Del 170, and Pul 0; Heat 750, Fil 4,Vel 40, Del 170, and Pul 200. The resulting nanopipette tips had innerdiameters ˜50 nm.

Cell Culture:

HeLa cells (maintained in the laboratory) were cultured (37° C., 5% CO₂)in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum, 1% sodium pyruvate and 1% Pen/Strep/Glu in 5% CO2 at 37° C. on aPetri dish covered with a thin layer of PDMS.

Reagents:

Poly-L-lysine (PLL; 19320-A) was purchased from Electron MicroscopySciences (Hatfield, Pa.). Dulbecco Modified Eagle's media (MT10017CV),fetal bovine serum (BW14502F), sodium pyruvate (BW13115E) andPen/Strep/Glu (SV3008201) were purchased from Fisher.Polydimethylsiloxane was purchase from Dow Corning, (Sylgard® 184silicon elastomer kit). (BSA, Qdots) PBS solutions at pH 7.4 wereprepared using standard methods. Aqueous reagents were prepared usingMilliQ water with >18M Ω·cm⁻¹ resistance.

Measurement Setup for Electroosmotic Ejection:

The setup consisted of a low-noise amplifier (Axopatch 200B, MolecularDevices, Sunnyvale, Calif.) for pipette bias and current measurement, amicromanipulator (MP-285) for coarse control in the X, Y, and Zdirections, a piezo actuator for fine control in the X, Y, and Zdirections, and a FPGA (National Instruments) for hardware control ofthe system. The system is further modified with a low-noise high voltagesource (Model 2100 Isolated Pulse Stimulator, A-M Systems), and a customrelay for switching between the low voltages required for feedbackcontrol, and the high voltages needed for electrophoretic materialejection. The system is controlled using custom coded software writtenin LabVIEW.

Example 2 Single Cell In Vivo Immunoassay with Nanopipette SensorsExperimental Section

Measurement Setup:

Since the current flowing through the nanopipette is too small topolarize a reference electrode, a two electrode setup was used. Thenanopipette sensor, acting as the working electrode, is backfilled withthe working buffer, and a Ag/AgCl electrode is inserted. Another Ag/AgClelectrode is placed in bulk solution acting as auxiliary/referenceelectrode. Both electrodes are connected to the Axopatch 700B amplifierwith the DigiData 1322A digitizer (Molecular Devices), and a PC equippedwith pClamp 10 software (Molecular Devices). The system remainedunstirred for the duration of the measurement, which was conducted atroom temperature.

Reagents: Poly-l-lysine (PLL; 19320-A) was purchased from ElectronMicroscopy Sciences (Hatfield, Pa.). Polyclonal antibody HPV16 E6 (C-19)and HPV18 E6 were purchased from Santa Cruz Biotechnology, Inc. (SantaCruz, Calif.). Dulbecco Modified Eagle's media (MT10017CV), fetal bovineserum (BW14502F), sodium pyruvate (BW13115E) and Pen/Strep/Glu(SV3008201) were purchased from Fisher. Polydimethylsiloxane waspurchased from Dow Corning, (Sylgard® 184 silicon elastomer kit). PBSsolutions at pH 7.4 were prepared using standard method. Aqueousreagents were prepared using MilliQ water with >18M Ω·cm⁻¹ resistance.

Nanopipette Sensor Fabrication:

Nanopipettes were fabricated from quartz capillaries with filaments,with an outer diameter of 1.0 mm and an inner diameter of 0.70 mm(QF100-70-5; Sutter Instrument Co.). The capillary was then pulled usinga P-2000 laser puller (Sutter Instrument Co.) programmed to fabricatenanopipettes with an inner diameter of ˜50 nm. Parameters used were:Heat 700, Fil 4, Vel 60, Del 150, and Pul 192. The resulting nanopipettetips had inner diameters ranging from 37 to 82 nm, with the meandiameter of 56 nm.

Antibody Immobilization:

Antibodies were immobilized through the following steps. First,nanopipettes were internally coated by filling with a 0.01% solution ofpoly-1-lysine in water, followed by centrifugation at 4600 rpm for 3min. The centrifugation step helps to get the solution to the very tipof the nanopipette. After the removal of excess PLL solution, thenanopipettes were baked at 120° C. for 1 h to stabilize the PLL coating.The nanopipette was then filled with a sulfo-SMCC solution (2 mg/ml, 10mM EDTA, 50 mM PBS), centrifuged at 4600 rpm for 3 min and thenincubated at room temperature for 1 h. Nanopipettes were then rinsedwith 0.01M PBS and centrifugated for at least 3 times to remove anyunreacted sulfo-SMCC molecules. Sulfo-SMCC contains an amine-reactiveN-hydroxysuccinimide (NHS ester) that reacts with the PLL amino groups,leaving a maleimide group available for the antibodies cross linkthrough a thioether bond. The nanopipettes were then incubated withantibody solution (10 μg/ml IgG, PBS, 1 h, 37° C.).Antibody-functionalized nanopipettes were then rinsed at least 3 timeswith PBS and centrifuged, to remove any unbound antibody and to providea smooth electrolyte filling throughout the tip.

Cell Lysis:

Hela cells were frozen into aliquots containing ˜10⁶ cells in growingmedia (10% DMSO). After thawing, cells were spun down to pellet, thesupernatant discarded and resuspended in 100 μL PBS solution. Covaris™,controlled by S-seried SonoLAB software, was used to lyse the cellmembrane. Conditions: Duty Cycle: 5%, Intensity 3, Cycles/Burst 200, for60 s. The cell lysate was centrifuged at 4000 rpm for 5 minutes toseparate biomolecules form cell debris. The vial was placed in ice andused within 2 hours to minimize protease inhibition.

Scanning Ion Conductance Microscope:

The SICM was built in-house and is based on a current amplifier(Molecular Instruments, Multiclamp 700B), a micromanipulator (SutterInstruments, MP-285) for coarse control, and a piezo actuator (PhyzikInstrumente, NanoCube) for fine control. The setup was controlled usinguser customized software (LabView) developed in-house specifically forthis application.

Cell Culture:

HeLa cells (maintained in the laboratory) were cultured (37° C., 5% CO₂)in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum, 1% sodium pyruvate and 1% Pen/Strep/Glu in 5% CO2 at 37° C. on aPetri dish covered with a thin layer of PDMS.

Intracellular Protein Detection

The present nanopipette sensors can selectively detect oncoproteins incells ex vivo. First, the selectivity of a nanopipette sensor fordistinguishing different HPV genotypes was investigated. The probesensor was functionalized with antibodies targeting HPV18E6 while thecontrol probe contained HPV16E6. HPV-18 E6 is known to be a “malignant”protein in humans. See, Li et al., “The human papilloma virus (HPV)-18E6 oncoprotein physically associates with Tyk2 and impairs Jak-STATactivation by interferon-alpha,” Oncogene 18(42):5727-5737 (1999). HPV16 E6 is described further at Gewin et al., “Identification of a noveltelomerase repressor that interacts with the human papillomavirustype-16 E6/E6-AP complex,” Genes Dev. 18(18):2269-2282 (2004). Bothsensors were immersed in a solution containing 100 pg/mL HPV18E6antigens. The specific protein-protein interaction instantly reduced thecurrent amplitude, through stepwise blockades, of the probe sensor (FIG.10). Their sequential binding at the close proximity of the nanopipettesensor opening alters the local impedance, thus inducing stepwisechanges in the recorded ionic current. These permanent blockades are notseen in the control sensor, whose electrical characteristic is notperturbed by the HPV18 protein E6 antigens. Furthermore, these blockadesare also distinguishable from those produced by molecular translocation,as protein molecules traversing the nanopipette generate shorter andtemporary blocks with a duration ranging in microseconds.

Next, the selectivity of the nanopipette sensor in cell lysate wastested. Serial dilutions were performed out of aliquots containing ˜10⁶HeLa cells to determine the sensitivity of the sensor. 350,000×dilutions does not result in any change in naked and HPV16E6functionalized nanopipettes while a 7% change in the measured currentwas detected for the HPV18E6 modified nanopipette sensor (FIG. 11).Control sensors still did not respond to ten times higher concentratedsamples whereas the measured current in the probe sensor dropped by 23%of its initial value. Results are summarized in Table 1.

TABLE 1 Normalized variation of the nanopipette sensor output current toserial dilution of 10⁶ HeLa cells lysate Dilutions Nanopipette HPV16E6sensor HPV18E6 sensor 350000X 100 100 93 35000X 100 100 77 35000x 93 869 3500x 2 4 4

Applied voltage was shown to play a very important role to limit if notavoid non specific adsorption, however this problem should always betaken into account as it becomes predominant in concentrated solutions,as FIG. 11 shows for dilution of the cell lysate of 3500× and belowwhere signal raised predominately form non specific adsorption. FIG. 11also shows the percentage change on the measured current after theaddition of HeLa cells lysate at different concentrations. The appliedvoltage was −500 mV in a PBS solution at pH 7.4.

Oncoprotein detection inside a single cell relies on a high precisionnanomanipulator and a current feedback loop to insert the nanopipettesensor to a defined depth into the probe cell. As a nanopipetteapproaches the surface of a cell the ionic current through the pipettewill decrease due to “current squeezing”, a well known effect, exploitedin scanning ion conductance microscopy. By monitoring the ionic currentthe precise position of the nanopipette can be determined and controlledwithin ˜200 nm of the cell membrane. Additionally, the feedbackmechanism ensures that the nanopipette tip does not come into contactwith anything but the cell membrane, preventing the tip from potentiallybreaking by coming into contact with other structures or substrates.HeLa cells were plated on a Petri dish coated with layer of PDMS andcovered with the complete medium.

The nanopipette sensor was biased at +500 mV when the electroosmoticflow is directed outwards from the nanopipette barrel. Thisconfiguration avoiding any cellular debris or molecules present in themedium to interact with the sensor and affect his performance. Thefeedback loop lowers the sensor with steps of 10 nm until the currentreduced to 92% of its initial value. After this initial positioningabove the cell membrane the nanopipette was inserted into the cell by 2μm at a speed of 100 μm/s. The sensor was inserted at a very high speedto reduce disturbance to the cell and maximize cell viability. Multiplepenetrations can be performed on the same cell, without any noticeablechange in the nanopipette current baseline and viability of the cell.Once inserted inside the cell, an AC voltage with amplitude of 500 mVand frequency of 5 Hz was applied to the nanopipette sensor to performthe sensing.

FIG. 12 shows the response of nanopipette sensors inserted into HeLacells. No noticeable change in the baseline current was detected uponinsertion of a bare nanopipette into HeLa cells while a 5% decrease wasmeasured with the control sensor functionalized with antibodiestargeting HPV16E6. HeLa cells are known to be infected only with HPV 18.This was confirmed by the penetration of HeLa cells with nanopipettesensors functionalized with anti HPV18E6. An 8% current drop wasmeasured after detection into HeLa cells due to the specific detectionof HPV18E6 oncoproteins. These results indicate that nanopipettetechnology can allow the study of protein-protein within individualcells and without any need for labeling.

Example 3 Cell Patterning by Controlled Electroosmotic Ejection UsingQuartz Nanopipettes

Patterns for cells were defined using polylysine and Bovine SerumAlbumin (BSA). 1 mg/ml laminin was used in the barrels of thenanopipette and deposited directly onto a PDMS substrate in a 35 mmPetri dish. Pattern deposition was confirmed by mixing quantum dots withthe electrolyte solution. Patterns could then be assessed usingfluorescence microscopy to visualize deposition, and check for errors inprinting. The substrate was then washed with 1 mg/ml BSA in a selfmasking step. After pattern definition the substrate was immersed incell culture media. HeLa cells were used as model cells in these studiesfor their ease of use and robustness. Cells were dispersed in media andallowed to settle onto the substrate. After several hours of incubationcells were gently rinsed with pre-warmed culture media to remove anycells not firmly attached to the substrate. Cell attachment was assessedboth qualitatively and quantitatively.

CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification areintended to convey details of methods and materials useful in carryingout certain aspects of the invention which may not be explicitly set outbut which would be understood by workers in the field. Such patents orpublications are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference andcontained herein, as needed for the purpose of describing and enablingthe method or material referred to.

1. An apparatus for manipulating individual cells on a substrate,comprising: (a) a multi-barreled nanopipette, having (i) a first barrelcontaining a first electrode arranged to be in contact with a liquid inthe first barrel; (ii) a second barrel adjacent the first barrelcontaining a second electrode arranged to be in contact with a liquid inthe second barrel; and (iii) an amplifier connected to said firstelectrode for controlling a voltage between said first electrode andsaid second electrode; (b) an xyz controller attached to saidmulti-barreled nanopipette for effecting mechanical movements of themulti-barreled nanopipette in submicron x and y steps, and effectingmovement of said multi-barreled nanopipette in a z direction towards oraway from the substrate, said xyz controller further having electroniccontrols for controlling said mechanical movements according to userdefined control; and (c) a circuit for controlling voltage between saidfirst electrode and said second electrode and for applying an ejectionvoltage to said first electrode to eject liquid from the first barrel ata desired location where liquid in said nanopipette is to be ejected andremoving said ejection voltage when the xyz controller effectsmechanical movement of the nanopipette away from the desired location,whereby the individual cell may be contacted with ejected liquid.
 2. Theapparatus of claim 1 wherein said circuit for controlling voltagecomprises a low noise amplifier for providing a pipette bias voltage andfor and current measurement of ionic current through the first barrel.3. The apparatus of claim 1 further comprising a piezoelectric actuatorfor submicron control of the xyz controller.
 4. The apparatus of claim 1further comprising a field-programmable gate array (FPGA) for user inputto determine said desired location.
 5. The apparatus of claim 4 whereinsaid FPGA is programmed for depositing spots in a pattern on saidsubstrate.
 6. The apparatus of claim 4 wherein said FPGA is programmedto inject a cell which is at a predetermined location on said substrate.7. The apparatus of claim 6 wherein said FPGA is programmed to inject anorganelle within said cell.
 8. The apparatus of claim 1 operativelyconnected to a substrate adapted to contain a plurality of individualcells, one cell each, in individual locations.
 9. The apparatus of claim8 wherein said substrate has cavities defined therein sized forreceiving an individual cell into a single cavity.
 10. The apparatus ofclaim 9 wherein the substrate comprises through-holes for applyingnegative pressure to hold a cell in a cavity.
 11. The apparatus of claim10 wherein the cavity comprises an electrode for attracting a cell tothe cavity.
 12. A method for depositing a liquid in a predefined patternhaving submicron features, comprising the steps of: (a) placing a liquidto be deposited into a multi-barreled nanopipette having (i) a firstbarrel containing a first electrode arranged to be in contact with aliquid in the first barrel; (ii) a second barrel adjacent the firstbarrel containing a second electrode arranged to be in contact with aliquid in the second barrel; and (iii) an amplifier connected to saidfirst electrode for controlling a voltage between said first and secondelectrodes; (b) actuating an xyz controller attached to saidmulti-barreled nanopipette for effecting mechanical movements of themulti-barreled nanopipette in submicron x and y steps, and effectingmovement of said multi-barreled nanopipette in a z direction towards oraway from the substrate, said xyz controller further having electroniccontrols for controlling said mechanical movements according to userdefined control; and (c) using a circuit for controlling voltage betweensaid first electrode and said second electrode and for applying anejection voltage to said first electrode to eject liquid from the firstbarrel at a desired location where liquid in said nanopipette is to beejected and removing said ejection voltage when the xyz controllereffects mechanical movement of the nanopipette away from the desiredlocation, whereby said liquid is deposited in said predeterminedpattern.
 13. The method of claim 12 wherein said liquid comprises a celladhesion material for allowing attachment of cells on a substrate onlyat areas of deposited cell adhesion material.
 14. The method of claim 12wherein said cell adhesion material is laminin.
 15. The method of claim12 wherein the substrate comprises a uniform polymer top surface. 16.The method of claim 12 wherein said liquid comprises a cell adhesionmaterial for allowing attachment of cells on the substrate only at areasof deposited cell adhesion material, and further comprising the step ofapplying to the substrate adherent cells to be cultured, whereby saidcells only adhere to regions having cell adhesion material.
 17. Themethod of claim 12 wherein regions having cell adhesion material are ata density of about at least 10 spots per 100 square micrometers.
 18. Themethod of claim 12 wherein the method is used to deposit differentmaterial from different barrels of the nanopipette.
 19. The method ofclaim 12 wherein the nanopipette is made of quartz and has an opening ofabout 20 to 100 nm.
 20. A method for injecting a material into a cell ona substrate, comprising the steps of: (a) placing a liquid to beinjected into a multi-barreled nanopipette, having (i) a first barrelcontaining a first electrode arranged to be in contact with a liquid inthe first barrel; (ii) a second barrel adjacent the first barrelcontaining a second electrode arranged to be in contact with a liquid inthe second barrel; and (iii) an amplifier connected to said firstelectrode for controlling a voltage between said first and secondelectrodes; (b) actuating an xyz controller attached to saidmulti-barreled nanopipette for effecting mechanical movements of themulti-barreled nanopipette in submicron x and y steps, and effectingmovement of said multi-barreled nanopipette in a z direction towards oraway from the substrate, said xyz controller further having electroniccontrols for controlling said mechanical movements according to userdefined control; and (c) using a circuit for controlling voltage betweensaid first electrode and said second electrode and for applying anejection voltage to said first electrode to eject liquid from the firstbarrel at a desired location where liquid in said nanopipette is to beejected and removing said ejection voltage when the xyz controllereffects mechanical movement of the nanopipette away from the desiredlocation, whereby said liquid is injected into said cell.
 21. The methodof claim 20 further comprising the step of immobilizing said cell onsaid substrate by placing said cell in a cavity in the substrate, saidcavity sized to hold only an individual cell.
 22. The method of claim 21further comprising the step of applying a pressure differential acrosssaid cavity to aid in immobilizing said cell.
 23. The method of claim 21wherein said material injected is selected from the group consisting ofa polynucleic acid, an antibody, and a dye.
 24. An apparatus fordetecting an analyte in an individual cell on a substrate, comprising:(a) a nanopipette having a barrel containing a first electrode, saidfirst electrode adapted to hold said analyte in contact with a second,reference electrode in a liquid contacting said cell and connected to acircuit for controlling a voltage between said first and secondelectrodes during use; and (b) an xyz controller attached to saidnanopipette for effecting mechanical movement of the nanopipette insubmicron x and y steps, and effecting movement of said nanopipette in az direction, said xyz controller further having electronic controls forcontrolling said mechanical movements according to user defined control;and (c) said nanopipette tip having an analyte binding materialimmobilized on an interior surface within said cell near a tip.
 25. Theapparatus of claim 24 wherein said analyte binding material is a proteinor a polynucleic acid.
 26. The apparatus of claim 24 wherein the analytebinding material is a protein linked by sulfo-SMCC to a PLL coating onsaid interior surface.
 27. A method to detect biomolecules in a singleliving cell comprising: (a) positioning a functionalized nanopipette toa defined depth into the cell; (b) monitoring the current to controlprecise position of nanopipette near the cell membrane; (c) insertingnanopipette at high speed into the cell; (d) applying high voltage; and(e) measuring current change to detect biomolecules in the cell whereinthe functionalized nanopipette is comprised in a system furthercomprising an amplifier for pipette bias and current measurement, amicromanipulator for control in the X, Y and Z directions, apiezo-actuator for fine control in the X, Y and Z directions, aconfigurable integrated circuit, a high voltage source, and a relay forswitching between low voltage and high voltage.
 28. The method of claim27 wherein the nanopipette is functionalized with antibodies specificfor the biomolecules.
 29. The method of claim 27 wherein the nanopipetteis functionalized with aptamers specific for the biomolecules.
 30. Themethod of claim 27 wherein the method is used in an immunoassay in asingle living cell.
 31. The method of claim 27 wherein the nanopipetteis functionalized with receptor ligands.
 32. The method of claims 27wherein said detecting is detecting oncoproteins in a single livingcell.